Paramagnetic particles that provide improved relaxivity

ABSTRACT

An improved contrast agent for magnetic resonance imaging comprises particles to each of which is coupled a multiplicity of chelating agents containing paramagnetic ions. In the improved agent, the position of the ion is offset from the surface of the particle so as to improve the relaxivity imparted by the contrast agent. A tether offsetting the chelate from the surface of the particle may optionally contain cleavage sites permitting more facile excretion of the chelated paramagnetic ion.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 10/153,395filed 21 May 2002 now U.S. Pat. No. 6,869,591 which claims benefit under35 U.S.C. § 119(e) to provisional application 60/368,100 filed 26 Mar.2002, and incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported in part by grants HL-59865 and CO-07121 from theNational Institutes of Health and from Philips Medical Systems, Best,Netherlands. The U.S. government has certain rights in this invention.

TECHNICAL FIELD

The invention relates to improved contrast agents for magnetic resonanceimaging (MRI). These agents are particles with paramagnetic ions offsetfrom the surface, optionally rendered cleavable therefrom.

BACKGROUND ART

Magnetic resonance imaging (MRI) has become a useful tool for diagnosisand for research. The current technology relies on detecting the energyemitted when the hydrogen nuclei in the water contained in tissues andbody fluids returns to a ground state subsequent to excitation with aradio frequency. Observation of this phenomenon depends on imposing amagnetic field across the area to be observed, so that the distributionof hydrogen nuclear spins is statistically oriented in alignment withthe magnetic field, and then imposing an appropriate radio frequency.This results in an excited state in which this statistical alignment isdisrupted. The decay of the distribution to the ground state can then bemeasured as an emission of energy, the pattern of which can be detectedas an image.

While the above described process is theoretically possible, it turnsout that the relaxation rate of the relevant hydrogen nuclei, left totheir own devices, is too slow to generate detectable amounts of energy,as a practical matter. In order to remedy this, the area to be imaged issupplied with a contrast agent, generally a strongly paramagnetic metal,which effectively acts as a catalyst to accelerate the decay, thuspermitting sufficient energy to be emitted to create a detectable brightsignal. To put it succinctly, contrast agents decrease the relaxationtime and increase the reciprocal of the relaxation time—i.e., the“relaxivity” of the surrounding hydrogen nuclei.

Two types of relaxation times can be measured. T₁ is the time for themagnetic distribution to return to 63% of its original distributionlongitudinally with respect to the magnetic field and the relaxivity ρ₁,is its reciprocal. T₂ measures the time wherein 63% of the distributionreturns to the ground state transverse to the magnetic field. Itsreciprocal is the relaxivity index ρ₂. In general, the relaxation timesand relaxivities will vary with the strength of the magnetic field; thisis most pronounced in the case of the longitudinal component.

Thus, a desirable characteristic of any contrast agents is to providethe signal with an enhanced relaxivity both for ρ₁ and ρ₂. The presentinvention provides such contrast agents.

It is also advantageous to facilitate the excretion of the paramagneticion, which may otherwise be toxic if it is retained in a subject. Thus,it would be advantageous to provide a mechanism for cleaving thechelated metal ion from the particles or from any lipid components thatmight result in cellular or liver uptake.

There is an extensive literature regarding contrast agents which arebased on chelated paramagnetic metals. For example, U.S. Pat. Nos.5,512,294 and 6,132,764 describe liposomal particles with metal chelateson their surfaces as MRI contrast agents. U.S. Pat. Nos. 5,064,636 and5,120,527 describe paramagnetic oil emulsions for MRI in thegastrointestinal tract. U.S. Pat. Nos. 5,614,170 and 5,571,498 describeemulsions that incorporate lipophilic gadolinium chelates, e.g.,gadolinium diethylene-triamine-pentaacetic acid-bis-oleate (Gd-DTPA-BOA)as blood pool contrast agents.

U.S. Pat. No. 5,804,164 describes water-soluble, lipophilic agents whichcomprise particularly designed chelating agents and paramagnetic metals.U.S. Pat. No. 6,010,682 and other members of the same patent familydescribe lipid soluble chelating contrast agents containing paramagneticmetals which are said to be able to be administered in the form ofliposomes, micelles or lipid emulsions.

Thus, in general, contrast agents may take the form of paramagneticmetals such as rare earth metals or iron mobilized in a form thatpermits substantial concentrations of the paramagnetic metal to bedelivered to the desired imaging area.

One method for providing useful concentrations of contrast agents hasbeen described by the present applicants in U.S. Pat. Nos. 5,780,010 and5,909,520. A nanoparticle is formed from an inert core surrounded by alipid/surfactant coating. The lipid/surfactant coating can then bemodified to couple the particle to a chelating agent containing aparamagnetic metal. In addition, the particle can be coupled to a ligandfor targeting to a specific site.

The present invention provides an improvement in the design of contrastagents whereby the relaxivity of the signal can be enhanceddramatically, and excretion can be facilitated.

DISCLOSURE OF THE INVENTION

The present invention concerns improved contrast agents with enhancedsignal relaxivities wherein this result is achieved by delivering theparamagnetic metal in high concentration in such a way as to provideincreased access to the hydrogen nuclei in the surrounding medium. Theagents of the invention employ particles, preferably, but notnecessarily, in a liquid emulsion, wherein the particles are coupled toa multiplicity of chelating agents, said chelating agents containing aparamagnetic ion. Rather than being coupled close to the surface, thechelate is offset from the surface of the particle so as to have betteraccess to the surrounding medium containing the hydrogen nuclei whichgenerate the signal. The offset is accomplished by use of a linkingmoiety; the linking moiety may optionally contain a cleavage site so asto permit removal of the chelate from the particles when desired. Theparticles may also contain ligands for targeting to specific sites, mayalso comprise drugs, and may be formed from fluorocarbons, thuspermitting ¹⁹F-MRI as a supplement.

Thus, in one aspect, the invention relates to a contrast agent formagnetic resonance imaging, which agent comprises particles, saidparticles coupled to a chelator containing a paramagnetic ion which ispositioned offset from the surface of the particles optionally by alinking moiety comprising a cleavage site, so as to provide theparamagnetic ion with substantial access to water molecules in asurrounding aqueous liquid.

In other aspects, the invention relates to methods to prepare the agentsof the invention and methods to use them in magnetic resonance imagingtechniques.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the size distribution of nanoparticles wherein a gadoliniumcomplex is coupled through phosphatidyl ethanolamine (Gd-DTPA-PE) andfor nanoparticles wherein a gadolinium chelate is coupled through bisoleate (Gd-DTPA-BOA).

FIG. 2 is a graph showing the dependence of relaxivity for thesurrounding hydrogen nuclei in units of (s*mM)⁻¹ for Gd-DTPA-BOA andGd-DTPA-PE as a function of magnetic field strength.

FIG. 3 shows the relaxivities of Gd-DTPA-BOA and Gd-DTPA-PE at 3° C. and37° C. as a function of Larmor frequency in megahertz. (The Larmorfrequency is the procession frequency of the energy-emitting nucleus.)

FIG. 4 shows sample ¹⁹F spectra at 0.47 T and 4.7 T magnetic fields.

FIG. 5 shows the effect of ¹⁹F concentration on ¹⁹F signal intensity inthe presence of emulsions of Gd-DTPA-BOA and Gd-DTPA-PE.

MODES OF CARRYING OUT THE INVENTION

The agents of the invention, useful in MRI, comprise particles to whicha multiplicity of chelating agents containing paramagnetic ions isbound. The particles will often form an emulsion or suspension in aliquid medium and can be delivered to the area to be imaged. As statedabove, the invention is directed to a method to improve relaxivity byoffsetting the chelating agent which contains the paramagnetic ion fromthe particle to which it is bound. This improvement and concept areapplicable to particle-borne chelating agents in general, includingliposomes, micelles, particles formed from lipoproteins, fullerenes,polymeric particles, such as latex, proteinaceous particles, orparticles formed from any other basic structure such as lipids,including oils and vitamins, carbohydrates, inorganic materials,particles designated as nanospheres or microspheres, and particles whichinclude gaseous forms such as microbubbles. The particles need not becomposed of a single component, but can include mixtures, for examplesynthetic oils, vitamins, halogenated chemicals, and the like. Anyparticulate carrier can serve as the carrier for compositions whichapply the methods of the invention.

In the particles of the invention, the coupling is such that theparamagnetic ion is offset from the surface of the particle at adistance, preferably, of at least 5 or 10 Å. Preferably the averagedistance at which the paramagnetic ion is found from the surface isbetween about 5–100 Å, preferably about 10–50 Å, and more preferablyabout 10–20 Å.

As used herein, the “surface” of the particle means the outer limit ofthe material comprising the particle at the location at which thechelator is coupled. Overall, the mean diameter of the particle itselfis compared to the mean distance from the center where the paramagneticions reside. This should be at least a 5 Å difference preferably atleast 10 Å.

The degree of offset can also be defined in terms of the resultantimpact on the relaxivity imparted by the offset. The imparted relaxivityis dependent on the strength of the magnetic field; the relaxivity on aper particle basis is, of course, determined in part by the number ofparamagnetic ions associated with the particle itself. At thearbitrarily chosen magnetic field strength of 0.47 T, the offset will besufficient to enhance the relaxivity on a per ion basis at least 1.2fold, preferably 1.5 fold, and more preferably 2.0 fold for ρ₁ and insimilar amounts for ρ₂. At the arbitrarily chosen magnetic field of 1.5T, the offsets will enhance these relaxivities by similar factors. At4.7 T, preferably the enhancement of ρ₁ is at least 1.5 fold, preferably2 fold and the enhancement of ρ₂ is at least two fold and preferablythree fold, again, on a per ion basis. In terms of units of relaxivityper se, the offset is such that the value for ρ₁ in (s*mM)⁻¹ at 0.47 Tis at least 20, and preferably 25, more preferably 30; at 1.5 T, thesevalues would be at least 20, and preferably 30, and at 4.7 T, at least10, and preferably 14. For ρ₂, the corresponding values at 0.47 T wouldbe at least 20, preferably 30, and more preferably 35; at 1.5 T, atleast 20, preferably 30; and at 4.7 T, at least 20, more preferably 40,and most preferably 60.

As applicants are able to apply to the particles a multiplicity ofchelators containing paramagnetic ions, considerably higher relaxivitiescan be obtained on a per particle basis. The fold increase in ρ₁ and ρ₂on a per particle basis is, of course, similar to that with respect tothe fold increase on a per ion basis. Applicants, however, have beenable to achieve values of ρ₁ in units of (s*mM)⁻¹ on a per particlebasis at 0.47 T, of at least 1.8×10⁶, preferably 2.0×10⁶, and morepreferably 2.5×10⁶. At 1.5 T, these values are similar and at 4.7 T,relaxivity values for ρ₁ are at least 8×10⁵, preferably 1×10⁶, morepreferably 1.1×10⁶.

For ρ₂ at 0.47 T, the relaxivity is preferably at least 2×10⁶, morepreferably 2.5×10⁶, and more preferably 3×10⁶ in these units. At 1.5 T,the values for ρ₂ are at least 1.6×10⁶, preferably 2.5×10⁶, and morepreferably 3×10⁶. At 4.7 T, ρ₂ is at least 3×10⁶, more preferably 4×10⁶,and more preferably 5×10⁶.

The offsetting is accomplished by spacing the dentate portion of thechelate through a tether to the surface of the particle. In oneembodiment, the surface is coated with a lipophilic material and thetether is anchored into the coating through a hydrophobic moiety such asone or more aliphatic hydrocarbon chains. In one preferred embodiment,the particles themselves can be described generally as nanoparticleshaving an inert core surrounded by a coating to which any desiredmaterials can be coupled. In the agent of the invention, these materialsmust include the chelate containing the paramagnetic ion.

With respect to these preferred particles, the inert core can be avegetable, animal or mineral oil, but is preferably a fluorocarboncompound—perfluorinated or otherwise rendered additionally inert.Mineral oils include petroleum derived oils such as paraffin oil and thelike. Vegetable oils include, for example, linseed, safflower, soybean,castor, cottonseed, palm and coconut oils. Animal oils include tallow,lard, fish oils, and the like. Many oils are triglycerides.

Fluorinated liquids are particularly useful as cores. These includestraight chain, branched chain, and cyclic hydrocarbons, preferablyperfluorinated. Some satisfactorily fluorinated, preferablyperfluorinated organic compounds useful in the particles of theinvention themselves contain functional groups. However, perfluorinatedhydrocarbons are preferred. The nanoparticle core may comprise a mixtureof such fluorinated materials. Typically, at least 50% fluorination isdesirable in these inert supports. Preferably, the inert core has aboiling point of above 20° C., more preferably above 30° C., still morepreferably above 50° C., and still more preferably above about 90° C.

Thus, the perfluoro compounds that are particularly useful in theabove-described nanoparticle aspect of the invention include partiallyor substantially or completely fluorinated compounds. Chlorinated,brominated or iodinated forms may also be used. A detailed list ofcompounds useful as nanoparticle cores is included after the Examplesbelow.

With respect to the coating on the nanoparticles in this aspect, therelatively inert core is provided with a lipid/surfactant coating thatwill serve to anchor the desired moieties to the nanoparticle itself. Ifan emulsion is to be formed, the coating typically should include asurfactant. Typically, the coating will contain lecithin type compoundswhich contain both polar and non-polar portions as well as additionalagents such as cholesterol. Typical materials for inclusion in thecoating include lipid surfactants such as natural or syntheticphospholipids, but also fatty acids, cholesterols, lysolipids,sphingomyelins, tocopherols, glucolipids, stearylamines, cardiolipins, alipid with ether or ester linked fatty acids, polymerized lipids, andlipid conjugated polyethylene glycol. Other surfactants are commerciallyavailable.

The foregoing may be mixed with anionic and cationic surfactants.

Fluorochemical surfactants may also be used. These includeperfluorinated alcohol phosphate esters and their salts; perfluorinatedsulfonamide alcohol phosphate esters and their salts; perfluorinatedalkyl sulfonamide alkylene quaternary ammonium salts;N,N-(carboxyl-substituted lower alkyl) perfluorinated alkylsulfonamides; and mixtures thereof. As used with regard to suchsurfactants, the term “perfluorinated” means that the surfactantcontains at least one perfluorinated alkyl group. A detailed list ofsurfactants, including fluorinated surfactants that can be used in thecoating, is found in the appendix after the Examples.

Typically, the lipids/surfactants are used in a total amount of 0.01–5%by weight of the nanoparticles, preferably 0.1–1% by weight. In oneembodiment, lipid/surfactant encapsulated emulsions can be formulatedwith cationic lipids in the surfactant layer that facilitate theadhesion of nucleic acid material to particle surfaces. Cationic lipidsinclude DOTMA, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoiumchloride; DOTAP, 1,2-dioleoyloxy-3-(trimethylammonio)propane; andDOTB,1,2-dioleoyl-3-(4′-trimethyl-ammonio)butanoyl-sn-glycerol may beused. In general the molar ratio of cationic lipid to non-cationic lipidin the lipid/surfactant monolayer may be, for example, 1:1000 to 2:1,preferably, between 2:1 to 1:10, more preferably in the range between1:1 to 1:2.5 and most preferably 1:1 (ratio of mole amount cationiclipid to mole amount non-cationic lipid, e.g., DPPC). A wide variety oflipids may comprise the non-cationic lipid component of the emulsionsurfactant, particularly dipalmitoylphosphatidylcholine,dipalmitoylphosphatidyl-ethanolamine or dioleoylphosphatidylethanolaminein addition to those previously described. In lieu of cationic lipids asdescribed above, lipids bearing cationic polymers such as polyamines,e.g., spermine or polylysine or polyarginine may also be included in thelipid surfactant and afford binding of a negatively charged therapeutic,such as genetic material or analogues there of, to the outside of theemulsion particles.

In addition to the above-described preferred embodiment, a multiplicityof other particulate supports may be used in carrying out the method ofthe invention. In other embodiments, for example, the particles may beliposomal particles. The literature describing various types ofliposomes is vast and well known to practitioners. As the liposomesthemselves are comprised of lipid moieties, the above-described lipidsand surfactants are applicable in the description of moieties containedin the liposomes themselves. These lipophilic components can be used tocouple to the chelating agent in a manner similar to that describedabove with respect to the coating on the nanoparticles having an inertcore. Micelles are composed of similar materials, and this approach tocoupling desired materials, and in particular, the chelating agentsapplies to them as well. Solid forms of lipids may also be used.

In another example, proteins or other polymers can be used to form theparticulate carrier. These materials can form an inert core to which alipophilic coating is applied, or the chelating agent can be coupleddirectly to the polymeric material through techniques employed, forexample, in binding affinity reagents to particulate solid supports.Thus, for example, particles formed from proteins can be coupled totether molecules containing carboxylic acid and/or amino groups throughdehydration reactions mediated, for example, by carbodiimides.Sulfur-containing proteins can be coupled through maleimide linkages toother organic molecules which contain tethers to which the chelatingagent is bound. Depending on the nature of the particulate carrier, themethod of coupling so that an offset is obtained between the dentateportion of the chelating agent and the surface of the particle will beapparent to the ordinarily skilled practitioner.

In all cases, to serve as MRI contrast agents, the particles are coupledthrough the required spacer to a chelator in which a transition metal isdisposed. Typical chelators are found in the patent documents cited inthe Background section above, and include porphyrins,ethylenediaminetetraacetic acid (EDTA),diethylenetriamine-N,N,N′,N″,N″-pentaacetate (DTPA),1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7 (ODDA), 16-diacetate,N-2-(azol-1(2)-yl)ethyliminodiacetic acids,1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid(DOTA),1,7,13-triaza-4,10,16-trioxacyclo-octadecane-N,N′,N″-triacetate(TTTA), tetraethylene glycols,1,5,9-triazacyclododecane-N,N′,N″,-tris(methylenephosphonic acid(DOTRP),N,N′,N″-trimethylammonium chloride (DOTMA) and analoguesthereof.

Suitable paramagnetic metals include a lanthanide element of atomicnumbers 58–70 or a transition metal of atomic numbers 21–29, 42 or 44,i.e., for example, scandium, titanium, vanadium, chromium, manganese,iron, cobalt, nickel, copper, molybdenum, ruthenium, cerium,praseodymium, neodymium, promethium, samarium, europium, gadolinium,terbium, dysprosium, holmium, erbium, thulium, and ytterbium, mostpreferably Gd(III), Mn(II), iron, europium and/or dysprosium.

According to the invention, the chelating moiety is coupled to theparticle through a spacer or tether which may be an aliphatic chain, apeptide, a polyethylene glycol polymer, or any suitable spacingmolecule. One end of the spacer is bound, preferably covalently, to thedentate portion of the chelating agent; the other is anchored to theparticle. The coupling to the particle can be covalent or the spacer maybe anchored through ionic bonding, hydrogen bonding or van der Waalsforces. When the particle surface comprises a lipid surface,particularly preferred anchoring moieties are the hydrocarbon sidechains of phosphatides or other di-substituted glycerol derivatives.

It is often advantageous to ensure that the spacer or tether iscleavable so that the paramagnetic ion chelate can be dissociated fromthe particle or from lipids that compose part of the particle. It may bedesirable to enhance excretion by liberating the chelate in ahydrophilic status to promote such excretion. Accordingly, the spacer ortether may contain one or more cleavage sites that either are activatedexternally, for example, by photoactivation, or which are continuouslyaccessed by enzymes present in the cells or bloodstream. Examples of theformer include specific linkages that are photoactivated, or cleaved byultrasound, as is understood in the art. After imaging has beencompleted, the nanoparticles are subjected to electromagnetic energy orultrasound as appropriate to effect cleavage. In the second instance,the spacer may be, or may include, peptides containing amino acidsequences that are susceptible to cleavage by circulating proteases ormay include polysaccharides, themselves susceptible to such cleavage.Any combination of such cleavage sites may be included. Thesusceptibility of the spacer or tether to cleavage thus enhancesexcretion and diminishes potential toxicity of the paramagnetic ion.

If continuous degradation is employed, the rate may be modulated byselecting spacers according to the available enzymatic activities and bysupplying a desired number of cleavage sites. However, it is well knownthat any peptide circulating in the bloodstream is ultimately destroyeddue to circulating proteases; similarly, polysaccharides are subject tocleavage by endogenous enzymes.

By appropriately coupling the chelating agents, substantial numbers ofchelators and paramagnetic ions can be coupled to the particles.Typically, the particles will be coupled to at least 10,000 chelatorsand/or paramagnetic ions, preferably 20,000 chelators and/orparamagnetic ions, more preferably 50,000 chelators and/or paramagneticions, more preferably at least 70,000 chelators and/or paramagnetic ionsand more preferably at least 100,000 chelators and/or paramagnetic ions.

As set forth above, the tether is such that an offset is obtainedsufficient to confer the relaxivity values described above, and spacingthe paramagnetic ion from the surface of the particle as described.

While the particles of the invention are required to comprise amultiplicity of paramagnetic ions coupled through chelating agents,additional components may also be coupled to these particles. Especiallyadvantageous for use of the contrast agents in some applications of MRIis the inclusion of a ligand which is a specific binding partner for atarget on a tissue desired to be imaged. It may also be desirable toprovide a biologically active substance and this may be included aswell.

Thus, in addition to the chelated paramagnetic metal ion, the particlesmay also be coupled to ligands for targeting and/or biologically activemolecules. It is possible also to include among the components coupledto the particles bearing the chelated paramagnetic ion, radionuclidesfor use in treatment or diagnosis.

Suitable biologically active materials include therapeutics such asantineoplastic agents, hormones, anticoagulants, and otherpharmaceuticals, representative examples of which are listed in theappendix after the Examples.

In one important embodiment of the invention, the particles containingthe offset contrast ion are targeted to a desired destination; however,this is not the case for all purposes. For example, the contrast agentsof the invention are useful in blood pool contexts or in thegastrointestinal tract where specific localization is unnecessary.However, the particles may also be targeted to specific organs or typesof tissue, including fibrin clots, liver, pancreas, neurons, or anytissue characterized by particular cell surface or other ligand-bindingmoieties. In order to effect this targeting, a suitable ligand iscoupled to the particle directly or indirectly. An indirect method isdescribed in U.S. Pat. No. 5,690,907, incorporated herein by reference.In this method, the lipid/surfactant layer of a nanoparticle isbiotinylated and the targeted tissue is coupled to a biotinylated formof its specific binding ligand. The biotinylated nanoparticle thenreaches its target through the mediation of avid in which couples thetwo biotinylated components.

In a preferred method, the specific ligand itself is coupled directly tothe particle, preferably but not necessarily, covalently. Thus, in such“direct” coupling, a ligand which is a specific binding partner for atarget contained in the desired location is itself linked to thecomponents of the particle, as opposed to indirect coupling where abiotinylated ligand resides at the intended target. Such direct couplingcan be effected through linking molecules or by direct interaction witha surface component. Homobifunctional and heterobifunctional linkingmolecules are commercially available, and functional groups contained onthe ligand can be used to effect covalent linkage. Typical functionalgroups that may be present on targeting ligands include amino groups,carboxyl groups and sulfhydryl groups. In addition, crosslinkingmethods, such as those mediated by glutaraldehyde could be employed. Forexample, sulfhydryl groups can be coupled through an unsaturated portionof a linking molecule or of a surface component; amides can be formedbetween an amino group on the ligand and a carboxyl group contained atthe surface or vice versa through treatment with dehydrating agents suchas carbodiimides. A wide variety of methods for direct coupling ofligands to components of particles in general and to components such asthose found in a lipid/surfactant coating in one embodiment are known inthe art. The foregoing discussion is non-comprehensive. In a specificcase which employs aptamers, it may be advantageous to couple theaptamer to the nanoparticle by the use of a cationic surfactant as acoating to the particles.

The targeting agent itself may be any molecule which is specific for anintended target. Commonly, such a ligand may comprise an antibody orportion thereof, an aptamer designed to bind the target in question, aknown ligand for a specific receptor such as an opioid receptor bindingligand, a hormone known to target a particular receptor, a peptidemimetic and the like. Certain organs are known to comprise surfacemolecules which bind known ligands; even if a suitable ligand isunknown, antibodies can be raised and modified using standard techniquesand aptamers can be designed for such binding.

Antibodies or fragments thereof are preferred targeting agents becauseof their capacity to be generated to virtually any target, regardless ofwhether the target has a known ligand to which it binds either nativelyor by design. Standard methods of raising antibodies, including theproduction of monoclonal antibodies are well known in the art and neednot be repeated here. It is well known that the binding portions of theantibodies reside in the variable regions thereof, and thus fragments ofantibodies which contain only variable regions, such as F_(ab), F_(v),and scF_(v) moieties are included within the definition of “antibodies.”Recombinant production of antibodies and these fragments which areincluded in the definition are also well established. If the imaging isto be conducted on human subjects, it may be preferable to humanize anyantibodies which serve as targeting ligands. Techniques for suchhumanization are also well known.

Thus, in summary, the contrast agents of the invention mandatorilycomprise particulate carriers which are coupled to a multiplicity ofchelating agents containing paramagnetic metal ions in such a mannerthat the paramagnetic metal ion is offset from the surface of theparticle so as more effectively to contact the surrounding mediumcontaining the hydrogen nuclei that emit signals under the conditions ofthe MRI image construction. The offset is such that the average distanceof the paramagnetic ion from the surface is of the order of 10 Å and atsuch a distance that the relaxivity of the surrounding hydrogen ions isenhanced, for example, at least 1.5 fold as compared to particleswherein the paramagnetic ion is directly attached to the surface,preferably enhanced 2-fold, and more preferably enhanced at least 2.5fold, and still more preferably enhanced at least 6 fold, or even 10fold. Alternatively, for example the offset distance from the surfacecan be judged on the basis of the ion-based relaxivity in (s*mM)⁻¹ as,e.g., for ρ₁ at least about 10, preferably 20 or 30 and up to 100 at amagnetic field of 1.5 T and ρ₂ at least about 20, preferably 30 or 40and up to 100 in these units at 1.5 T; or the relaxivity on a perparticle basis at least, for example, about 0.5×10⁶, preferably 1.5×10⁶and up to 15×10⁶; (s*mM)¹ ⁻at 1.5 T for ρ₁ and at least about 1.0×10⁶preferably 3.0×10⁶ and up to 15×10⁶ in these units for ρ₂. As statedabove, in addition to the offset paramagnetic ions, the particles mayalso contain targeting moieties, bioactive agents, or radionuclides.Preferably, targeting ligands are included.

It is understood that with respect to any material comprised by theparticles, a multiplicity of copies may be included. For the chelatorcontaining a paramagnetic ion, typically, the particles contain at least2,000 copies, typically at least 5,000, more typically at least 10,000or 100,000 or 500,000. For targeting agents, only one or two, or severalor more copies may be included. Variable numbers of drug molecules maybe contained.

The precise process for preparation of the contrast agents of theinvention is variable, and depends on the nature of the particulatecarrier and the choice of tether or spacer molecules. As describedabove, solid particles which contain reactive groups can be coupleddirectly to the tether or spacer; lipid-based particles such as oilemulsions, solid lipids, liposomes, and the like, can include lipophilicmaterials containing reactive groups which may covalently, then, becoupled to linking moieties which bear the dentate portion of thechelating agent. In one particularly preferred embodiment, the processinvolves mixing a liquid fluorocarbon compound that forms the core of ananoparticle and the components of a lipid/surfactant coating for thatparticle in an aqueous suspension, microfluidizing, and, if desired,harvesting and sizing the particles. The components to be coupled can beincluded in the original mixture by virtue of their initial coupling toone or more components of the lipid/surfactant coating, or the couplingto additional moieties can be conducted after the particles are formed.

A typical preparation of one preferred agent of the invention isdescribed as follows:

The emulsion comprises perfluorocarbon (e.g., perfluorooctylbromide 40%w/v, PFOB), a surfactant co-mixture (2.0%, w/v) and glycerin (1.7%, w/v)in aqueous medium. The surfactant co-mixture may includedipalmitoylphosphatidyl choline, cholesterol, dipalmitoylphosphatidylethanolamine-DTPA-Gd (or may include, for example,phosphoethanolamine-N-4 PEG₍₂₀₀₀₎-(p-maleimidophenyl)butyramide(MPB-PEG-PE) if further coupling to a targeting ligand is required)phosphatidylethanolamine, and/or sphingomyelin in varying molar ratios,which are dissolved in chloroform/methanol, evaporated under reducedpressure, dried in a 50° C. vacuum oven overnight and dispersed intowater by sonication. Optionally, one or more therapeutic agents may beincluded. The suspension is combined with the perfluorooctaylbromide anddistilled, added to deionized water, blended and then emulsified at20,000 PSI for three minutes (S110 Microfluidics microemulsification).

For targeting, a thiolated peptidomimetic ligand is coupled to themaleimide derivatized phospholipid included in the coating in 50 mMphosphate, 10 mM EDTA buffer at pH 6.65 overnight under a nitrogenatmosphere. Alternatively, phosphoethanolamine-N-4PEG₍₂₀₀₀₎-p-maleimidophenyl)butyramide (MPB-PEG-PE) may be dried into alipid film under vacuum and the thiolated peptidomimetic ligand may becoupled to the lipid upon resuspension with in 50 mM phosphate, 10 mMEDTA buffer at pH 6.65 so as to be included in the particles uponformation.

Alternatively the ligand, such as an antibody, antibody fragment orsmall molecule analogue thereof (e.g., ScF_(v)) may be reacted withN-succinimidyl S-acetylthioacetate (SATA) for 30 min, dialyzedovernight, deprotected with hydroxylamine, dialyzed in oxygen freebuffers, then coupled to the nanoparticles at room temperature for 2hours. A control emulsion is prepared identically with a nonderivatizedphosphatidylethanolamine substituted into the surfactant commixture andthe ligand conjugation steps are omitted.

Vialed peptidomimetic emulsions are heat sterilized with neutral pHadjustment (NaCO₃) at 121° C. for 15 min. Nanoparticles for conjugationto antibodies are heat sterilized before coupling and ligand conjugationis completed under aseptic conditions in a laminar flow biohood. Theimproved nanoparticle-based contrast agents are then useful in obtainingmagnetic resonance images in subjects using standard techniques forobtaining such images.

The contrast agents may be used without targeting ligands for obtainingimages where homing to a site is unnecessary, such as blood pool images.However, where specific organs are to be imaged, targeted forms of theparticles are preferred.

The use of perfluoro carbons as the basis for the nanoparticles in thisembodiment of the invention is further advantageous in that resonanceimages of the ¹⁹F contained in the particle can also be concomitantlyobtained and serve to verify the translocation of the contrast agent tothe desired locations in the subject.

The following examples are intended to illustrate but not to limit theinvention.

Preparation A Nanoparticle Preparation

Paramagnetic nanoparticles were produced in a modification of theprocedure described by Lanza, G, et al., Circulation (1996)94:3334–3340. Briefly, the emulsions comprised 40% (v/v)perfluorooctylbromide (PFOB; MMM, St. Paul, Minn.), 2% (w/v) saffloweroil, 2% (w/v) of a surfactant co-mixture, 1.7% (w/v) glycerin and waterrepresenting the balance. The surfactant co-mixture included 63 mole %lecithin (Avanti Polar Lipids, Inc., Alabaster, Ala.), 15 mole %cholesterol (Sigma Chemical Co., St. Louis, Mo.), 2 mole %dipalmitoyl-phosphatidylethanolamine (Avanti Polar Lipids, Inc.,Alabaster, Ala.), and 20 mole % of the paramagnetic lipophilic chelate.The lipophilic chelate was either gadoliniumdiethylene-triamine-pentaacetic acid-bis-oleate (Gd-DTPA-BOA; GatewayChemical Technologies, St. Louis, Mo.) or DTPA-phosphatidylethanolamine(DTPA-PE; Gateway Chemical Technologies, St. Louis, Mo.). The surfactantcomponents were dissolved in chloroform, evaporated under reducedpressure, dried in a 50° C. vacuum oven overnight and dispersed intowater by sonication. The suspension was pre-emulsified in a blender withPFOB, safflower oil and distilled deionized water for 30 to 60 secondsand then emulsified in a M110S Microfluidics emulsifier (Microfluidics,Newton, Mass.) at 20,000 PSI for four minutes. The completed formulationwas placed in crimp sealed vials and blanketed with nitrogen. Particlesizes were determined in triplicate at 37° C. with a laser lightscattering submicron particle sizer (Malvern Instruments, Malvern,Worcestershire, UK).

EXAMPLE 1 Preparation of Contrast Agent

As set forth in Preparation A, Either gadoliniumdiethylene-triamine-pentaacetic acid-bis-oleate (Gd-DTPA-BOA; GatewayChemical Technologies, St. Louis, Mo.) or DTPA-phosphatidylethanolamine(DTPA-PE; Gateway Chemical Technologies, St. Louis, Mo.), was includedin the surfactant co-mixture at a concentration of 20 mole % of thetotal lipid membrane. Gadolinium chloride was added in excessproportions as a post-emulsification step to nanoparticles formulatedwith DTPA-PE. Unbound gadolinium was removed by dialysis on thenanoparticles against distilled deionized water (300,000 MW cut-off,Spectrum Laboratories, Rancho Dominguez, Calif.). Gadolinium-DTPA-BOAwas incorporated into the surfactant lipids as the complete paramagneticcompound. Both Gd-DTPA-BOA and Gd-DTPA-PE emulsions were tested for freeGd³⁺ using the arsenazo III reaction and showed no sign of unboundlanthanide.

The concentration of Gd³⁺ was calculated from the reactants used duringformulation, while the concentration of nanoparticles was derived fromthe nominal particle size (i.e. particle volume of a sphere) and theamount of perfluorocarbon formulated into the preparation. The number ofGd³⁺-complexes per nanoparticle was determined from the ratio of theconcentrations of Gd³⁺ and nanoparticles in the emulsion.

The nominal particle sizes and distributions of the Gd-DTPA-PE andGd-DTPA-BOA nanoparticles were similar and overlapping, as shown inFIG. 1. Table 1 shows additional properties:

TABLE 1 Properties of Paramagnetic Nanoparticles. Gd-DTPA-BOA Gd-DTPA-PEParticle Size (nm) 287 261 Polydispersity Index 0.28 0.23 [Gd³⁺] (mM)3.36 5.79 Gd³⁺ Ions/Particle 56,900 73,600 [Particles] (nM) 59.1 78.7

Each lipophilic nanoparticle presented more than 50,000 Gd-complexesalong the water-lipid interface. The capacity of these nanoparticles tosupport high paramagnetic payload is important to the efficacy of theseagents when employed for molecular imaging of biochemical epitopes.

EXAMPLE 2 Paramagnetic Nanoparticle Sample Preparation and Assessment ofT₁ and T₂ Relaxivities at 0.47 T.,1.5 T and 4.7 T

Gd-DTPA-BOA and Gd-DTPA-PE nanoparticles prepared in Example 1 werediluted to 0, 4, 6, 8, 10 and 12% PFOB (v/v) with distilled deionizedwater. The initial nanoparticle formulation contained 26.1 mol/L ¹⁹F andthe diluted aliquots had 0, 3.915, 5.22, 6.525 and 7.83 mol/L ¹⁹F,respectively. Total gadolinium content was determined by neutronactivation analysis. The gadolinium contents of the Gd-DTPA-BOAnanoparticle dilutions were 0; 0.336; 0.504; 0.672; 0.84; and 1.01mmol/L Gd³⁺. The paramagnetic ion concentrations in Gd-DTPA-PE sampleswere 0; 0.579; 0.869; 1.16; 1.45; and 1.74 mmol/L Gd³⁺.

The proton longitudinal and transverse relaxation rates (1/T₁ and I/T₂,respectively) of each sample were measured at 40° C. on a Bruker MQ20Minispec NMR Analyzer with a field strength of 0.47 T. T₁ was measuredusing an inversion recovery sequence with 10 inversion delay values,while T₂ was measured with a Carr-Purcell-Meiboom-Gill (CPMG) sequence.The T₁ and T₂ relaxivities (i.e., ρ₁ and ρ₂, respectively) werecalculated from the slope of the linear least-squares regression oflongitudinal and transverse relaxation rates versus Gd³⁺ (i.e., ionrelaxivity) or nanoparticle (i.e., particle relaxivity) concentrationsand are reported in units of (s*mM)⁻¹.

Spin echo images from a clinical scanner (Gyroscan NT, PowerTrak 6000,Philips Medical Systems, Best, Netherlands) obtained with a standard 11cm diameter surface coil were used to measure the relaxivity of the twonanoparticle formulations at 1.5 T. A six chamber phantom allowed allsix dilutions to be studied in parallel. To accommodate the differentrelaxation times of the two paramagnetic formulations, different imagingparameters were applied. T₁ was calculated from an inversion recoveryMRI pulse sequence. The measurement for the Gd-DTPA-BOA phantom includedsix inversion times (TV) ranging from 50 to 1500 ms, while theGd-DTPA-PE value utilized seven T₁ values ranging from 5 ms to 200 ms.The signal intensity (S1) from each chamber was fit to the equation:S1_(T1) =S1₀*(1−EXP(−T1/T ₁)),   [1]where S1₀ represents the equilibrium signal intensity. The T₂ value forGd-DTPA-BOA was derived from a multi-echo sequence with 8 echo times(TE) ranging from 20 ms to 160 ms. Nine separate images with echo timesranging from 4.5 ms to 200 ms were used to calculate the T₂ relaxationfor the Gd-DTPA-PE phantom. MRI signal intensity was fit to theequation:S1_(TE) =S1₀ *EXP(−TE/T ₂).   [2]The imaging parameters common for both formulations were: TR=1000 ms,TE=5 ms (unless otherwise noted), number of signal averages=4, imagematrix=128 by 128, FOV=7 cm by 7 cm, flip angle=90°, slice thickness=5mm.

The relaxivities of the two paramagnetic formulations were also measuredwith a 4.7 T magnet interfaced to a Varian INOVA console (VarianAssociates, Palo Alto, Calif.) using a 5 cm birdcage coil. As statedearlier, a six chamber phantom was used to study the various emulsiondilutions concurrently. T₁ and T₂ values were obtained with inversionrecovery (TE=7.2 ms, T₁ varied from 1 to 800 ms) and spin echo (TEvaried from 7.2 to 100 ms) pulse sequences, respectively. The imageswere collected with TR=3000 ms, number of signal averages=4, imagematrix=256 by 256, FOV=4 cm by 4 cm, flip angle=90°, slice thickness=2mm.

Finally, the relaxivities of the two paramagnetic preparations weremeasured independently at magnetic fields ranging from 0.05 T to 1.3 T(2–56 MHz) using a custom built variable field relaxometer (SouthwestResearch Institute, San Antonio, Tex.). The samples were measured attemperatures of 3° and 37° C. A saturation recovery pulse sequence with32 incremental τ values was used to measure ρ₁, while ρ₂ was measuredusing a CPMG pulse sequence with 500 echoes and a 2 ms inter-echo delaytime.

Table 2 shows T₁ and T₂ relaxivities of the Gd-DTPA-BOA and Gd-DTPA-PEparamagnetic formulations determined at three magnetic field strengths.

TABLE 2 Relaxivities of Gd-DTPA-BOA and Gd-DTPA-PE emulsions at threedifferent field strengths. Ion-Based Particle-Based Relaxivity (s*mM)⁻¹Relaxivity (s*mM)⁻¹ Magnetic Paramagnetic Field Chelate ρ₁ ρ₂ ρ₁ ρ₂ 0.47T Gd-DTPA-BOA 21.3 ± 0.2 23.8 ± 0.3 1,210,000 ± 10,000 1,350,000 ±20,000 Gd-DTPA-PE 36.9 ± 0.5 42.3 ± 0.6 2,710,000 ± 40,000 3,110,000 ±50,000  1.5 T Gd-DTPA-BOA 17.7 ± 0.2 25.3 ± 0.6 1,010,000 ± 10,0001,440,000 ± 30,000 Gd-DTPA-PE 33.7 ± 0.7 50 ± 2 2,480,000 ± 50,000 3,700,000 ± 100,000  4.7 T Gd-DTPA-BOA  9.7 ± 0.2 29.4 ± 0.3  549,000 ±9,000 1,670,000 ± 20,000 Gd-DTPA-PE 15.9 ± 0.1   80 ± 0.7 1,170,000 ±6,000  5,880,000 ± 50,000

At all magnetic field strengths, both the ion-based and particle-basedρ₁ of the Gd-DTPA-PE formulation were about two-fold greater (p<0.05)than ρ₁ of the Gd-DTPA-BOA agent. Similarly, ion-based andparticle-based ρ₂ of the Gd-DTPA-PE agent were approximately two-foldhigher (p<0.05) than ρ₂ of the Gd-DTPA-BOA system at the lowest magneticfield strength (0.47 T), and this relative difference was more thanthree-fold greater (p<0.05) at the highest field strength (4.7 T).

At 1.5 T, a typical medical imaging field strength, the ion-based ρ₁ andρ₂ for Gd-DTPA-BOA were 17.7±0.2 (s*mM)⁻¹ (mean±standard error) and25.3±0.6 (s*mM)⁻¹, respectively, consistent with our previous reportedestimates (Flacke, S., et al., Circulation (2001) 104:1280–1285).Incorporation of Gd-DTPA-PE (as opposed to Gd-DTPA-BOA) increased theion-based ρ₁ and ρ₂ to 33.7±0.7 (s*mM)⁻¹ and 50.0±2 (s*mM)⁻¹,respectively. More importantly from a targeted agent perspective, theparticle-based ρ₁ and ρ₂ for Gd-DTPA-BOA were 1,010,000±10,000 (s*mM)⁻¹and 1,440,000±30,000 (s*mM)⁻¹, respectively, and for Gd-DTPA-PEnanoparticles the particle-based ρ₁ and ρ₂ were 2,480,000±50,000(s*mM)⁻¹ and 3,700,000±100,000 (s*mM)⁻¹, respectively. To our knowledge,particulate or molecular relaxivities in these ranges are the highestvalues reported to date for any targeted or blood pool paramagneticcontrast agent at these field strengths.

The influence of magnetic field strength on relaxivity is shown in FIG.2. The magnitudes of ion and particle longitudinal relaxivities declinedas magnetic field strength increased from 0.47 T to 4.7 T, whereas theion and particle transverse relaxivities progressively increased withhigher field strengths. Although the particle longitudinal relaxivitydeclined about 50% at 4.7 T compared to 1.5 T, the particle ρ₁ remainedvery high. As a ligand-targeted contrast agent, the decreases inrelaxivity at higher field strengths will be effectively offset byreduced voxel sizes, smaller partial volume dilution effects andimproved signal to noise.

Variable field relaxometry measurements showed that ρ₁ of both emulsionswas dominated by the long correlation time (τ_(c)) of the slowlytumbling emulsion complex (FIG. 3). In fact, the particles wererelatively so large, that there was almost no field dependence(dispersion). In contrast, the ρ₂ values initially followed those of ρ₁but did not decrease at higher fields in accordance with expectationsbased on the Solomon-Bloembergen equations (Wood, M. L., J. Mag. Res.Imag. (1993) 3:149–156) (due to the non-dispersive term involvingτ_(c)). For the Gd-DTPA-BOA emulsion, the “peak” ρ₁ relaxivity wasaround 25 (s*mM)⁻¹ and the maximum value of ρ₂ was 30 (s*mM)⁻¹. Thevalue of ρ₁ was largely independent of temperature, but ρ₂ increased atthe lower temperature. For the Gd-DTPA-PE emulsion, however, therelaxivities were much higher, with ρ₁ reaching 40 (s*mM)⁻¹ at 40 MHz(approx. 1.7 T) and ρ₂ reaching 50 (s*mM)⁻¹ at 56 MHz (1.3 T). Thetemperature dependence of Gd-DTPA-PE was also different from Gd-DTPA-BOAwith ρ₁ decreasing at the lower temperature and ρ₂ remaining independentof temperature. The relaxometry values were consistent with analogousmeasurements made at 0.47 T and 1.5 T (Table 2). Moreover, thetemperature dependence of these curves suggested that the Gd-DTPA-PEchelate has better access to water (i.e., faster exchange) compared toGd-DTPA-BOA.

EXAMPLE 3 ¹⁹F Spectroscopy and Imaging

The ¹⁹F signal intensities of Gd-DTPA-BOA and Gd-DTPA-PE nanoparticleswere characterized at 0.47 T and 4.7 T, but the necessary RF channel wasunavailable for study at 1.5 T. At 0.47 T, ¹⁹F spectra were collectedfrom each sample and the signal was quantified with respect to areagent-grade PFOB standard. At 4.7 T, spin echo ¹⁹F images werecollected from a six chamber phantom using a 1.5 cm single turn solenoidcoil, dual-tuned to ¹H and ¹⁹F. The imaging parameters were: TR=5000 ms,TE=6.3 ms, number of signal averages=35, image matrix=256 by 256, FOV=2by 2 cm, flip angle=90°, slice thickness=1 mm. The relative ¹⁹F signalintensity of each chamber was determined from the image pixel grayscaleusing Scion Image (version: beta 3b) (Scion Corporation, Frederick,Md.).

Representative fluorine spectra collected at 0.47 T and 4.7 T (FIG. 4)from the PFOB nanoparticle formulations revealed a markedly improvedspectral resolution, as expected, at the higher field strength, whichallows the multiple resonances of PFOB to be clearly separated. Bycomparison, these multiple resonance peaks collapsed into a singleunsymmetrical resonance at 0.47 T equivalent to the integration of allPFOB resonances with improved signal to noise ratio. The ¹⁹F signalintensity of paramagnetic nanoparticles increased linearly withconcentration at 0.47 T and 4.7 T independent of the lipophilicgadolinium chelate employed (FIG. 5). At 0.47 T, ¹⁹F signal intensitiesat each concentration of the two paramagnetic formulations werevirtually superimposable, implying that the PFOB contents were nearlyequivalent. At 4.7 T, ¹⁹F signal intensity estimates of the twoparamagnetic nanoparticle formulations were more variable butstatistically identical. The increased variation in measurements at the4.7 T field strength was due to errors in signal intensity estimationsecondary to chemical shift artifacts. Despite these issues, theamplitude of the fluorine signal was directly correlated withnanoparticle concentration.

APPENDIX Typical Components

Typical Core Components

Among the perfluorocarbon compounds which may be employed areperfluorotributylamine (FC47), perfluorodecalin (PP5),perfluoromethyldecalin (PP9), perfluorooctylbromide,perfluorotetrahydrofuran (FC80), perfluroether (PID), [(CF₃)₂ CFOCF₂(CF₂)₂ CF₂ OCF (CF₃)₂]perfluoroether (PIID) [(CF₃)₂ CFOCF₂ (CF₂)₆ CF₂OCF (CF₃)₂], perfluoroetherpolymer (Fomblin Y/01), perfluorododecane,perfluorobicyclo[4.3.0.]nonane, perfluorotritrimethylbicyclohexane,perfluorotripropylamine, perfluoroisopropyl cyclohexane,perfluoroendotetrahydrodicyclopentadiene, perfluoroadamantane,perfluoroexotetrahydrodicyclopentadiene, perfluorbicyclo[5.3.0.]decane,perfluorotetramethylcyclohexane,perfluoro-1-methyl-4-isopropylcyclohexane, perfluoro-n-butylcyclohexane,perfluorodimethylbicyclo[3.3.1.]nonane, perfluoro-1-methyl adamantane,perfluoro-1-methyl-4-t butylcyclohexane, perfluorodecahydroacenapthane,perfluorotrimethylbicyclo[3.3.1.]nonane, perfluoro-1-methyl adamantane,perfluoro-1-methyl-4-t butylcyclohexane, perfluorodecahydroacenaphthene,perfluorotrimethylbicyclo[3.3.1.]nonane, perfluoro-nundecane,perfluorotetradecahydrophenanthrene,perfluoro-1,3,5,7-tetramethyladamantane, perfluorododecahydrofluorene,perfluoro-1-3-dimethyladamantane, perfluoro-n-octylcyclohexane,perfluoro-7-methyl bicyclo[4.3.0.]nonane,perfluoro-p-diisopropylcyclohexane, perfluoro-m-diisopropylcyclohexane,perfluoro-4-methyloctahydroquinolidizine,perfluoro-N-methyldecahydroquinoline, F-methyl-1-oxadecalin,perfluorooctahydroquinolidizine, perfluoro 5,6-dihydro-5-decene,perfluoro-4,5-dihydro-4-octene, perfluorodichlorooctane andperfluorobischlorobutyl ether, perfluorooctane, perfluorodichlorooctane,perfluoro-n-octyl bromide, perfluoroheptane, perfluorodecane,perfluorocyclohexane, perfluoromorpholine, perfluorotripropylamine,perfluortributylamine, perfluorodimethylcyclohexane,perfluorotrimethylcyclohexane, perfluorodicyclohexyl ether,perfluoro-n-butyltetrahydrofiuran, and compounds that are structurallysimilar to these compounds. Chlorinated perfluorocarbons, such aschloroadamantane and chloromethyladamantane as described in U.S. Pat.No. 4,686,024 may be used. Such compounds are described, for example inU.S. Pat. Nos. 3,962,439; 3,493,581, 4,110,474, 4,186,253; 4,187,252;4,252,824; 4,423,077; 4,443,480; 4,534,978 and 4,542,147.

Surfactants

Commercially available surfactants are Pluronic F-68, Hamposyl™ L30 (W.R. Grace Co., Nashua, N.H.), sodium dodecyl sulfate, Aerosol 413(American Cyanamid Co., Wayne, N.J.), Aerosol 200 (American CyanamidCo.), Lipoproteol™ LCO (Rhodia Inc., Mammoth, N.J.), Standapol™ SH 135(Henkel Corp., Teaneck, N.J.), Fizul™ 10–127 (Finetex Inc., ElmwoodPark, N.J.), and Cyclopol™ SBFA 30 (Cyclo Chemicals Corp., Miami, Fla.);amphoterics, such as those sold with the trade names: Deriphat™ 170(Henkel Corp.), Lonzaine™ JS (Lonza, Inc.), Niranol™ C2N-SF (MiranolChemical Co., Inc., Dayton, N.J.), Amphoterge™ W2 (Lonza, Inc.), andAmphoterge™ 2WAS (Lonza, Inc.); non-ionics, such as those sold with thetrade names: Pluronic™ F-68 (BASF Wyandotte, Wyandotte, Mich.),Pluronic™ F-127 (BASF Wyandotte), Brij™ 35 (ICI Americas; Wilmington,Del.), Triton™ X-100 (Rohm and Haas Co., Philadelphia, Pa.), Brij™ 52(ICI Americas), Span™ 20 (ICI Americas), Generol™ 122 ES (Henkel Corp.),Triton™ N-42 (Rohm and Haas Co.), Triton™ N-101 (Rohm and Haas Co.),Triton™ X-405 (Rohm and Haas Co.), Tween™ 80 (ICI Americas), Tween™ 85(ICI Americas), and Brij™ 56 (ICI Americas) and the like.

Also included may be egg yolk phospholipids, alkylphosphoryl choline oralkylglycerolphosphoryl choline surfactants, and specific examples ofthese such as 1,2-dioctylglycero-3-phosphoryl choline,1,2-ditetradecylglycero-3-phosphoryl choline,1,2-dihexadecylglycero-3-phosphoryl choline,1,2-dioctadecylglycero-3-phosphorylcholine,1-hexadecyl-2-tetradecylglycero-3-phosphoryl choline,1-octadecyl-2-tetradecylglycero-3-phosphoryl choline,1-tetradecyl-2-octadecylglycero-3-phosphoryl choline,1-hexadecyl-2-octadecylglycero-3-phosphorylcholine,1–2-dioctadecylglycero-3-phosphoryl choline,1-octadecyl-2-hexadecylglycero-3-phosphoryl choline,1-tetradecyl-2-hexadecylglycero-3-phosphoryl choline,2,2-ditetradecyl-1-phosphoryl choline ethane and1-hexadecyltetradecylglycero-3-phosphoryl choline.

Suitable perfluorinated alcohol phosphate esters include the free acidsof the diethanolamine salts of mono- andbis(1H,1H,2H,2H-perfluoroalkyl)phosphates. The phosphate salts,available under the trade name “Zonyl RP” (E. I. Dupont de Nemours andCo., Wilmington, Del.), are converted to the corresponding free acids byknown methods. Suitable perfluorinated sulfonamide alcohol phosphateesters are described in U.S. Pat. No. 3,094,547. Suitable perfluorinatedsulfonamide alcohol phosphate esters and salts of these includeperfluoro-n-octyl-N-ethylsulfonamidoethyl phosphate,bis(perfluoro-n-octyl-N-ethylsulfonamidoethyl) phosphate, the ammoniumsalt ofbis(perfluoro-n-octyl-N-ethylsulfonamidoethyl)phosphate,bis(perfluoro-decyl-N-ethylsulfonamidoethyl)-phosphateand bis(perfluorohexyl-N ethylsulfonamidoethyl)-phosphate. The preferredformulations use phosphatidylcholine,derivatized-phosphatidylethanolamine and cholesterol as the aqueoussurfactant.

Illustrative Bioactive Agents

Biologically active molecules which may be included and coupled to thecoating include antineoplastic agents, such as platinum compounds (e.g.,spiroplatin, cisplatin, and carboplatin), methotrexate, fluorouracil,adriamycin, mitomycin, ansamitocin, bleomycin, cytosine arabinoside,arabinosyl adenine, mercaptopolylysine, vineristine, busulfan,chlorambucil, melphalan (e.g., PAM, L-PAM or phenylalanine mustard),mercaptopurine, mitotane, procarbazine hydrochloride dactinomycin(actinomycin D), daunorubicin hydrochloride, doxorubicin hydrochloride,taxol, mitomycin, plicamycin (mithramycin), aminoglutethimide,estramustine phosphate sodium, flutamide, leuprolide acetate, megestrolacetate, tamoxifen citrate, testolactone, trilostane, amsacrine(m-AMSA), asparaginase (L-asparaginase) Erwina asparaginase, etoposide(VP-16), interferon α-2a, interferon α-2b, teniposide (VM-26),vinblastine sulfate (VLB), vincristine sulfate, bleomycin, bleomycinsulfate, methotrexate, adriamycin, arabinosyl, hydroxyurea,procarbazine, and dacarbazine; mitotic inhibitors such as etoposide andthe vinca alkaloids, radiopharmaceuticals such as radioactive iodine andphosphorus products; hormones such as progestins, estrogens andantiestrogens; anti-helmintics, antimalarials, and antituberculosisdrugs; biologicals such as immune serums, antitoxins and antivenins;rabies prophylaxis products; bacterial vaccines; viral vaccines;aminoglycosides; respiratory products such as xanthine derivativestheophylline and aminophylline; thyroid agents such as iodine productsand anti-thyroid agents; cardiovascular products including chelatingagents and mercurial diuretics and cardiac glycosides; glucagon; bloodproducts such as parenteral iron, hemin, hematoporphyrins and theirderivatives; biological response modifiers such as muramyldipeptide,muramyltripeptide, microbial cell wall components, lymphokines (e.g.,bacterial endotoxin such as lipopolysaccharide, macrophage activationfactor), sub-units of bacteria (such as Mycobacteria, Corynebacteria),the synthetic dipeptide N-acetyl-muramyl-L-alanyl-D-isoglutamine;anti-fungal agents such as ketoconazole, nystatin, griseofulvin,flucytosine (5-fc), miconazole, amphotericin B, ricin, cyclosporins, andβ-lactam antibiotics (e.g., sulfazecin); hormones such as growthhormone, melanocyte stimulating hormone, estradiol, beclomethasonedipropionate, betamethasone, betamethasone acetate and betamethasonesodium phosphate, vetamethasone disodium phosphate, vetamethasone sodiumphosphate, cortisone acetate, dexamethasone, dexamethasone acetate,dexamethasone sodium phosphate, flunisolide, hydrocortisone,hydrocortisone acetate, hydrocortisone cypionate, hydrocortisone sodiumphosphate, hydrocortisone sodium succinate, methylprednisolone,methylprednisolone acetate, methylprednisolone sodium succinate,paramethasone acetate, prednisolone, prednisolone acetate, prednisolonesodium phosphate, prednisolone tebutate, prednisone, triamcinolone,triamcinolone acetonide, triamcinolone diacetate, triamcinolonehexacetonide, fludrocortisone acetate, oxytocin, vassopressin, and theirderivatives; vitamins such as cyanocobalamin neinoic acid, retinoids andderivatives such as retinol palmitate, and α-tocopherol; peptides, suchas manganese super oxide dismutase; enzymes such as alkalinephosphatase; anti-allergic agents such as amelexanox; anti-coagulationagents such as phenprocoumon and heparin; circulatory drugs such aspropranolol; metabolic potentiators such as glutathione; antitubercularssuch as para-aminosalicylic acid, isoniazid, capreomycin sulfatecycloserine, ethambutol hydrochloride ethionamide, pyrazinamide,rifampin, and streptomycin sulfate; antivirals such as acyclovir,amantadine azidothymidine (AZT, DDI, Foscamet, or Zidovudine), ribavirinand vidarabine monohydrate (adenine arabinoside, ara-A); antianginalssuch as diltiazem, nifedipine, verapamil, erythritol tetranitrate,isosorbide dinitrate, nitroglycerin (glyceryl trinitrate) andpentaerythritol tetranitrate; anticoagulants such as phenprocoumon,heparin; antibiotics such as dapsone, chloramphenicol, neomycin,cefaclor, cefadroxil, cephalexin, cephradine erythromycin, clindamycin,lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin,dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin,nafcillin, oxacillin, penicillin including penicillin G and penicillinV, ticarcillin rifampin and tetracycline; antiinflammatories such asdiflunisal, ibuprofen, indomethacin, meclofenamate, mefenamic acid,naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac,tolmetin, aspirin and salicylates; antiprotozoans such aschloroquine,hydroxychloroquine, metronidazole, quinine and meglumineantimonate; antirheumatics such as penicillamine; narcotics such asparegoric;opiates such as codeine, heroin, methadone, morphine andopium; cardiac glycosides such as deslanoside, digitoxin, digoxin,digitalin and digitalis; neuromuscular blockers such as atracuriummesylate, gallamine triethiodide, hexafluorenium bromide, metocurineiodide, pancuronium bromide, succinylcholine chloride (suxamethoniumchloride), tubocurarine chloride and vecuronium bromide; sedatives(hypnotics) such as amobarbital, amobarbital sodium, aprobarbital,butabarbital sodium, chloral hydrate, ethchlorvynol,ethinamate,flurazepam hydrochloride, glutethimide, methotrimeprazinehydrochloride, methyprylon, midazolam hydrochloride, paraldehyde,pentobarbital, pentobarbital sodium, phenobarbital sodium, secobarbitalsodium, talbutal, temazepam and triazolam; local anesthetics such asbupivacaine hydrochloride, chloroprocaine hydrochloride, etidocainehydrochloride, lidocaine hydrochloride, mepivacaine hydrochloride,procaine hydrochloride and tetracaine hydrochloride; general anestheticssuch as droperidol, etomidate, fentanyl citrate with droperidol,ketamine hydrochloride, methohexital sodium and thiopental sodium; andradioactive particles or ions such as strontium, iodide rhenium andyttrium.

1. A contrast agent for magnetic resonance imaging (MRI), which agentcomprises particles, wherein said particles are coupled to a chelatorcontaining a paramagnetic ion which ion is positioned offset from thesurface of the particles by a tether, so as to provide said ionsubstantial access to hydrogen nuclei in a surrounding liquid, wherebythe relaxivity of said nuclei is enhanced, wherein said particles aremicroparticles or nanoparticles comprised of an inert core comprising aperfluorocarbon compound, an animal oil, a vegetable oil, a mineral oil,a protein or a polymer surrounded by a lipid/surfactant coating, andwherein said chelator is positioned offset from the surface of theparticles through said tether which tether is covalently bound to saidchelator and to at least one component of the particles, and wherein thetether contains at least one site susceptible to cleavage.
 2. The agentof claim 1, wherein said offset positions said ion at a mean distance ofat least 5 Å from the surface of the particle.
 3. The agent of claim 1,wherein said offset is such that the particle provides a ρ₁ of at leastabout 0.5×10⁶(s*mM)⁻¹ or a ρ₂ of at least about 1×10⁶(s*mM)⁻¹ at a fieldstrength of 1.5 T on a per particle basis.
 4. The agent of claim 1,wherein said offset is such that the particle provides a ρ₁ of at leastabout 10(s*mM)⁻¹ or a ρ₂ of at least about 20(s*mM)⁻¹ at a fieldstrength of 1.5 T on a per ion basis.
 5. The agent of claim 1, whereinsaid offset is such that ρ₁ is increased at least about 1.5-fold or ρ₂is increased at least about 1.5-fold at a field strength of 1.5 T on aper particle basis as compared to ρ₁ or ρ₂ of particles wherein theparamagnetic ion resides at less than 5 Å from the surface.
 6. The agentof claim 1, wherein said tether comprises at least one peptide linkage.7. The agent of claim 1, wherein said cleavage site is activated byelectromagnetic radiation or ultrasound.
 8. The agent of claim 1,wherein the particles are contained in a liquid emulsion.
 9. The agentof claim 1, wherein said inert core comprises a perfluorocarboncompound.
 10. The agent of claim 1, wherein the inert core comprises amixture of at least one fluorocarbon and at least one oil.
 11. The agentof claim 1, wherein the chelator is selected from the group consistingof a porphyrin, ethylenediaminetetraacetic acid (EDTA),diethylenetriamine-N,N,N′,N″,N″-pentaacetate (DTPA),1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diacetate,N-2-(azol-1(2)-yl)ethyliminodiacetic acid,1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′-tetraacetic acid,1,7,13-triaza-4, 10, 16-trioxacyclo-octadecane-N,N′,N′′-triacetate,tetraethylene glycol,1,5,9-triazacyclododecane-N,N′,N″,-tris(methylene)phosphonic acid, andN,N′,N″-trimethylammonium chloride.
 12. The agent of claim 11, whereinthe chelator is DTPA.
 13. The agent of claim 1, wherein the paramagneticion is selected from the group consisting of scandium, titanium,vanadium, chromium, manganese, iron, cobalt, nickel, copper, molybdenum,ruthenium, cerium, praseodymium, neodymium, promethium, samarium,europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, andytterbium.
 14. The agent of claim 13, wherein the paramagnetic ion isgadolinium.
 15. The agent of claim 1, wherein the lipid/surfactantcoating comprises at least one compound selected from the groupconsisting of a natural phospholipid, a synthetic phospholipid, a fattyacid, a cholesterol, a lysolipid, a sphingomyelin, a tocopherol, aglucolipid, a stearylamine, a cardiolipin, a lipid with an ether-linkerfatty acid, a lipid with an ester linked fatty acid, a polymerizedlipid, and a polyethylene glycol-conjugated lipid.
 16. The agent ofclaim 1, wherein said particles are coupled to at least 10,000 chelatorsper particle.
 17. The agent of claim 1, wherein said particles furthercomprise a coupled target-specific ligand.
 18. The agent of claim 17,wherein said target specific ligand is an antibody, an antibodyfragment, a peptide, an aptamer, a peptide mimetic, a drug or a hormone.19. The agent of claim 18, wherein said target specific ligand is anantibody or fragment of an antibody.
 20. The agent of claim 19, whereinsaid antibody is humanized and/or is a single chain F_(v) antibody. 21.The agent of claim 17, wherein said particles comprise at least about 2copies of said target-specific ligand per particle.
 22. The agent ofclaim 17, wherein said target-specific ligand is coupled directly tosaid particles.
 23. The agent of claim 1, wherein said particles furthercomprise a biological agent.
 24. A method for magnetic resonance imaging(MRI), which method comprises administering the agent of claim 1 to asubject, permitting said agent to accumulate at a site of said subjectfor which an image is desired; and detecting an image of said sitegenerated by hydrogen nuclei at said site.
 25. The method of claim 24,wherein said site comprises a specific binding partner for a ligand, andwherein said particles further are coupled to a ligand specific for saidspecific binding partner.
 26. The method of claim 24, which furthercomprises effecting cleavage at said site susceptible to cleavage.
 27. Amethod for magnetic resonance imaging (MRI), which method comprisesadministering the agent of claim 9 to a subject, permitting said agentto accumulate at a site of said subject for which an image is desired;and detecting an image of said site generated by hydrogen nuclei at saidsite; which further comprises detecting an image generated by ¹⁹ Fcontained in said particles at said site.
 28. The agent of claim 1,wherein the inert core comprises a fluorocarbon liquid or an oil. 29.The method of claim 26, wherein the cleavage is enzymatic or photoactivecleavage.